Current systems and methods for collection, extraction, and detection of nucleic acid from biological samples for testing are typically complicated, requiring multiple steps with technically trained personnel, and not optimized for processing samples with large volumes or for preventing cross-contamination in sample processing and stabilization for shipment.
A variety of body fluids, such as blood, plasma, serum, Cerebrospinal fluid (CSF), pleural effusion, ascites, urine etc., contain short chain nucleic acid (NA) fragments, namely, cell-free nucleic acids (cfNA), or circulating nucleic acids (cNA). Altered nucleic acids, originated endogenously from a tumor, or “exogenously” from fetus or pathogenic infection inside the body, may present as cfNA in the peripheral blood at very low concentrations and may be detectable, and further, be distinguishable from normal host cfNA. Extraction of sufficient amount of those cfNA from plasma or serum for testing requires processing a relatively large volume of fluid, which imposes a unavoidable technical challenge in clinical diagnostic settings. Accordingly, there is a need the field for new methods to meet such challenges.
Exemplary such methods for detecting, for example, tuberculosis, are described in Pending PCT published application WO2012135815, invented by the inventor of this application, and incorporated herein by reference. Such testing, however, may be most useful in regions of the world lacking ready access to the expensive processing equipment used in analysis of the samples. Accordingly, there is a need in the art for a collection system and methodology that will permit capturing nucleic acid in sufficient amounts from large volume biological samples, to run later analysis, to prevent contamination from the environment and operators, and to preserve and ship the nucleic acid, so that nucleic acid can be collected at a point of care facility using relatively inexpensive equipment, and then shipped in a stabilized form to a central location for further processing and assays.
Various methods of extraction suitable for isolating circulating DNA or RNA from large volumes of biological fluids are known, such as those described, for example, in QIAamp® Circulating Nucleic Acid Handbook, (2nd edition, 02, 2011, Qiagen), and an improved spin column extraction method described in U.S. Pat. No. 5,234,809 (Boom technology). U.S. Pat. No. 5,346,994 describes a technology an organic liquid extraction method using phenol-chloroform. Both of these methods may be used for large volume extraction, such as from plasma or serum specimens, but the organic reagents are toxic, which limits its use.
U.S. Pat. Nos. 7,897,378 and 8,158,349 describe devices and method for purifying or isolating nucleic acids from larger sample volumes, including systems comprising a pair of cooperating hollow bodies through which samples are passed into a collection vessel, with nucleic acids bound to a binding material in one of the hollow bodies. The hollow body containing the retained sample is transferred to a first receiving vessel for washing, then the purified or isolated nucleic acids are eluted and collected in a second receiving vessel for further analysis.
U.S. Pat. No. 5,234,809 (Boom), for example, incorporated herein by reference, discloses a method for isolating nucleic acids, which is suitable for a multiplicity of different uses. It describes a method for isolating nucleic acids from nucleic acid-containing starting materials by incubating said starting material with a chaotropic buffer and a DNA-binding solid phase. The chaotropic buffers effect, if necessary, both lysis of the starting material and binding of the nucleic acids to the solid phase.